Frederick Baker (Graduate Student)
Hannah Neiswender (Research Associate)
Rajalakshmi Veeranan Karmegam (Research Associate).
94-98 BA Carthage College, Kenosha, WI
98-04 PhD. Medical College of Wisconsin, Milwaukee, WI
04-07 Case Western Reserve University, Cleveland, OH
07-09 University of North Carolina, Chapel Hill, NC
The Gonsalvez lab is currently funded by an R01 grant from the National Institutes of General Medical Sciences (2013-2018). Past funding include a grant from the American Cancer Society and an intramural grant from Augusta University.
Research Goals: Most eukaryotic cells are highly polarized and require polarity for proper function. Consequently, a contributing factor to a wide spectrum of human diseases is defective cell polarity. Our lab examines the mechanisms by which cells establish and maintain polarity.
How do cells establish polarity? The key to establishing polarity is to sort certain proteins to specific sites within the cell. For instance, the apical surface of an epithelial cell contains a different set of proteins than those found at the basolateral surface. Similarly, the proteins found within the axon of a neuron are often different from those founds at the tips of dendrites. The cell accomplishes this task of protein sorting using several mechanisms.
mRNA localization. Many cell types in the human body establish polarity via a process known as mRNA localization. This process enables the cell to restrict the synthesis of proteins to a particular region within the cell. The model organism that we use for these studies is Drosophila melanogaster (fruit fly). In particular, our lab examines the localization of oskar mRNA in Drosophila oocytes. oskar mRNA is an ideal candidate for investigation because the factors involved in its localization also function to localize mRNAs in mammalian cells. However, the Drosophila model provides us with the unique opportunity to study the process of mRNA localization in the context of the whole organism, rather than using isolated cells in culture. In addition, the wide array of genetic and molecular tools available for use with Drosophila will facilitate deeper and more rapid discovery. We have recently demonstrated that localization of oskar mRNA requires the concerted activities of two microtubule motor proteins, Kinesin and Dynein. An important next question is to determine the mechanism by which these motors associate with oskar mRNA.
The top panel in the above figure shows a control egg chamber. The motor, Dynein, as well as oskar mRNA are localized at the posterior pole. The bottom panel represents an egg chamber in which Dynein has been depleted. This results in the delocalization of oskar mRNA.
Endocytosis. Endocytosis is the mechanism by which cargoes are internalized and sorted within the cell. In addition to mRNA localization, endocytic trafficking also contributes to the establishment of cell polarity. Our lab has recently shown that the Dynein motor complex performs an essential function in endocytic maturation. When Dynein is inhibited, internalized cargo is trapped in enlarged endocytic vesicles (larges circles in the top panel). Our current efforts are focused on determining the precise mechanism by which Dynein functions in the endocytic pathway.
The top panel of this figure illustrates the large endocytic intermediates that are observed when Dynein function is compromised (large circles). The bottom panel show electron microscopy images from control (left) and Dynein inhibited (right) egg chambers.
In order for cargo to be trafficked through the endocytic pathway, the membrane on endocytic vesicles has to become curved. This is a thermodynamically unfavorable process. Protein containing a domain known as a BAR domain are known to aid in this process. BAR domain proteins generate either membrane tubules or protrusions when over-expressed in cells. Our lab is currently studying the functions of a protein called Sh3px1. This protein contains a unique kind of BAR domain. When Sh3px1 is over-expressed in cells, it dramatically alters cell morphology. Some cells form long tubules, whereas other cells form long membrane protrusions. We are currently exploring the role of Sh3xp1 in development and tissue morphogenesis.
The top panel of this figure shows a control Drosophila S2 cells that is expressing GFP. In the middle panel, the cell is over-expressing GFP-Sh3px1. This cell has formed long tubules. The bottom panel shows a cell over-expressing GFP-Sh3px1. This cell has formed long protrusions.
Long term goals: Defects in cell polarity are associated with several diseases including cancer, polycystic kidney disease and Charcot-Marie-Tooth disorder. The long-term goal of our research is to design better therapies to treat these debilitating diseases by determining how healthy cells establish polarity, and by understanding the way in which defective molecular pathways contribute to the disease state.
Approaches: We use a variety of tools such as genetics, cell biology and biochemistry to test our hypotheses. Numerous molecular techniques such as RT-PCR, western blotting, immunoprecipitation and in situ hybridization are routinely used. Studies are performed using Drosophila, Drosophila cell lines and mammalian cell lines.
Lawrence Hicks, Guojun Liu, Ukken Fiona, Sumin Lu, Kathryn E. Bollinger, Kate O’Connor-Giles and Graydon B. Gonsalvez. Depletion or over-expression of Sh3px1 results in dramatic changes in cell morphology. 2015. Biol Open. In press.
Guojun Liu, Paulomi Sanghavi, Kathryn E. Bollinger, Libby Perry, Brendan Marshall, Penny Roon, Tsubasa Tanaka, Akira Nakamura, and Graydon B. Gonsalvez. 2015. Efficient Endocytic Uptake and Maturation in Drosophila Oocytes Requires Dynamitin/p50. Genetics 201: 631-649.
Paulomi Sanghavi, Shobha Laxani, Xuan Li, Simon Bullock and Graydon B. Gonsalvez. Dynein associates with oskar mRNPs and is required for their efficient net plus-end localization in Drosophila oocytes. PLoS One. 2013 Nov 11;8(11):e80605.
Graydon B. Gonsalvez and A. Gregory Matera. Postranslational modification of Sm proteins: Diverse roles in snRNP assembly and germline specification. RNA Processing in Eukaryotes (ed J. Wu), Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany. 2013.
Graydon B. Gonsalvez and Roy M. Long. Spatial regulation of translation through RNA localization. F1000 Biol Rep. 2012;4:16. doi: 10.3410/B4-16. Epub 2012 Aug 1.
Paulomi Sanghavi, Sumin Lu, and Graydon B. Gonsalvez. A functional link between localized Oskar, dynamic microtubules, and endocytosis. Dev Biol. 2012 Jul 1;367(1):66-77.
Graydon B. Gonsalvez, T.K. Rajendra, Ying Wen and A. Gregory Matera. Sm proteins specify germ cell fate by regulating the localization of cytoplasmic oskar mRNA. Development, July 2010 137: 2341-2351.
Graydon B. Gonsalvez, Kavita Praveen, Amanda Hicks, Liping Tian, and A. Gregory Matera. Sm protein methylation is dispensable for snRNP assembly in Drosophila melanogaster. RNA. 2008, May 14:878-887.
Graydon B. Gonsalvez, Liping Tian, Jason K. Ospina, Francois-Michel Boisvert, Angus I. Lamond, and A. Gregory Matera. Two distinct arginine methyltransferases are required for biogenesis of Sm-class ribonucleoproteins. Journal of Cell Biology. 2007 Aug 27;178(5):733-40.
T.K. Rajendra, Graydon B. Gonsalvez, Karl B. Schpargel, Helen K. Salz, and A. Gregory Matera. A Drosophila model for Spinal Muscular Atrophy reveals a sarcomeric function for SMN in striated muscle. Journal of Cell Biology. 2007 Mar 12;176(6):831-41.
Carl R. Urbinati, Graydon B. Gonsalvez, John P. Aris, and Roy M. Long. Loc1p is required for efficient assembly and nuclear export of the 60S ribosomal subunit. Mol Genet. Genomics. 2006 Oct;276(4):369-77.
Graydon B. Gonsalvez, T.K. Rajendra, Liping Tian, and A. Gregory Matera. The Sm-protein methyltransferase, dart5, is essential for germ-cell specification and maintenance. Current Biology. 2006 Jun 6; 16(11): 1077-1089
Jason K. Ospina, Graydon B. Gonsalvez, Janna Bednenko, Edward Darzynkiewicz , Larry Gerace, and A. Gregory Matera. Crosstalk between Snurportin1 Subdomains. M ol Biol Cell . 2005 Oct;16(10):4660-71.
Graydon B. Gonsalvez, Carl Urbinati, and Roy M. Long (2005). RNA Localization in Yeast - Moving Towards a Mechanism. Biology of The Cell. Jan; 97(1):75-86
Graydon B. Gonsalvez, Jamie L. Little and Roy M. Long (2004). ASH1 mRNA Anchoring Requires Reorganization of the Myo4p/She3p/She2p Transport Complex. Journal of Biological Chemistry. Oct 29; 279(44):46286-94
Graydon B. Gonsalvez, Katrina A. Lehmann, Derek K. Ho, Eleni Stanitsa , James R.Williamson, and Roy M. Long (2003) . She2p mRNA-Binding Activity is Required for Proper Asymmetric Sorting of the Myo4p-She3p Complex to daughter cells. RNA, Nov;9(11):1383-99.
Roy M. Long, Wei Gu, Xiuhua Meng, Graydon Gonsalvez, Robert H. Singer, and Pascal Chartrand. (2001). An Exclusively Nuclear RNA-binding Protein Affects Asymmetric Localization of ASH1 mRNA and Ash1p in Yeast. Journal of Cell Biology, 153 : 307-318.